Hepatocyte growth factor (HGF) is discovered as a potent proliferation promoting factor for mature hepatocytes, and is a protein whose gene has been cloned by gene cloning (Biochem Biophys Res Commun, 122, 1450 (1984); Proc. Natl. Acad. Sci. USA, 83, 6489, (1986); FEBS Letter, 22, 231 (1987); Nature, 342, 440 (1989); Proc. Natl. Acad. Sci. USA, 87, 3200 (1991)). Subsequent studies have been proving that HGF functions as a liver regenerating factor in vivo for restoring and regenerating a damaged liver, and also has an angiogenic effect, playing a significant role in treating or prevention of ischemic and arterial diseases (Symp. Soc. Exp. Biol., 47 cell behavior, 227-234 (1993); Proc. Natl. Acad. Sci., 90; 1937-1941 (1993); Circulation, 97, 381-390 (1998)). In other words, it was reported that significant angiogenesis was observed, and the blood flow was improved, reduction of the blood pressure was inhibited and symptoms of ischemia were improved as a result, when HGF is administered to a rabbit model of hindlimb ischemia. Because of such reports, it is considered today that HGF expresses and functions as one of angiogenic factors.
Thus, HGF has various functions including that of an angiogenic factor, and different attempts have been made for utilizing it as a drug However, the half-life of HGF in the blood has been a problem. The half-life of HGF is as short as about 10 min so that it was difficult to maintain a concentration in the blood, and transportation of an effective dose to an affected region was also a problem.
In the case of protein preparations in general, it is commonly known that intravenous administration is often employed. Regarding administration of HGF to the ischemic disease model described above, examples of intravenous and intra-arterial administration, for example, were illustrated (Circulation, 97, 381-390 (1998)). Although the effectiveness of HGF to ischemic or arterial diseases have been shown, no conclusion has been made specifically with regard to an effective method of administration or dose of HGF. In the case of HGF, in particular, because there are such problems of the half-life and transportation to an affected region as described, no conclusion has been made regarding an effective method of administration or dose of HGF.
It is an object of the invention to provide preparations to be administered for treating or preventing ischemic diseases or arterial diseases which contain HGF as an active ingredient. More particularly, the invention relates to preparations administered intramuscularly for treating or preventing ischemic diseases or arterial diseases of the heart or extremities by administration to an affected part and local muscular region around it. The preparation for intramuscular administration according to the invention is directly administered intramuscularly to an ischemic region of the heart or extremities that represents a damaged region, for example, and an efficiency of utilization of HGF administered is very high, so that a dose can be reduced in comparison with the case of intravenous administration. In addition, because transpiration, distribution and effect from the region to which the preparation is administered to the blood and the whole body excluding muscular tissues and peripheral tissues of the region is low, it is effective in such manner that an effect to other organs can be reduced, and it provides a higher selectivity.
For adapting HGF to ischemic or arterial diseases, the inventors studied hard about a dose and a method of administration. Such methods as intravenous bolus administration, intravenous continuous administration, subcutaneous administration, intraperitoneal administration and intramuscular administration have been considered and studied as methods of administration of HGF for the above-described diseases.
As described above, in the case of protein preparation in general, it is commonly known that the intravenous administration is often employed, and it has been a common knowledge that it is effective to allow a drug to act from inside a blood vessel, because ischemic and arterial diseases are angiopathy. Consequently, as for administration of HGF to ischemic and arterial disease models, intravenous or intra-arterial administration was employed in most examples, information including literature about intramuscular administration of HGF was scarce, and no report on intracorporeal kinetics of HGF could be found. Thus, no report was found regarding an attempt of intramuscular administration of HGF to an ischemic or arterial disease model, and hitherto it has never been clarified, therefore, whether intramuscular administration of HGF is effective or not.
The inventors studied pharmacokinetics of HGF administered intramuscularly to an ischemic region of hindlimb in comparison with that administered through another route (intravenous administration) by using rats. As a result, in the case of the intramuscular administration, it was found that HGF administered was maintained at a very high concentration in the region to which it was administered, and transportation to serum, liver and kidney was low compared to the case of intravenous administration. Additionally, similarly to the case of intramuscular administration, when HGF was subcutaneously administered, HGF was maintained at a high concentration in the region to which it was administered, and transportation from the region to the circulating blood was low.
On the other hand, in the case HGF was administered intravenously, it was found that HGF was at a low concentration in a muscle, while it was maintained at a high concentration in the blood. Accordingly, it was recognized for the first time that an efficiency was low in the intravenous administration, and it was effective to directly administer HGF intramuscularly to an affected region or an area thereof, when it was intended to allow HGF to act to an ischemic region and a muscle or a blood vessel present therein.
Further, as a result of comparison between intramuscular administration and intravenous bolus administration of HGF, it was found that the maximum concentration in the blood, liver and kidney in the case of intramuscular administration was a hundredth or less, more preferably a thousandth or less of that in the intravenous bolus administration. Moreover, it was found that the concentration in muscular tissues of an administration region in the case of intramuscular administration was 50 times or more, more preferably 200 times or more of that in the intravenous bolus administration.
Furthermore, as a result of comparison of the AUC in the blood, liver and kidney between intramuscular administration and intravenous bolus administration, it was found that the AUC in the blood, liver and kidney in the case of intramuscular administration was a fifth or less, more preferably a tenth or less of that in the intravenous bolus administration. In addition, the AUC in muscular tissues of an administration region in the case of intramuscular administration was 50 times or more, more preferably 200 times or more of that in the intravenous administration.
As described, because it was found that HGF was maintained at a high concentration in an administration region of affected area, it was succeedingly examined whether HGF in the muscular tissues was transported to tissues and blood vessels in the affected area, and really provided its efficacy as HGF. In other words, the function of HGF was confirmed by using, as an index, presence or absence of phosphorylation of c-Met that is a receptor of HGF. As a result, when HGF of 30 to 3000 μg/kg was administered intramuscularly to rats, it was found that a Tyr residue of c-Met (a receptor of HGF) was phosphorylated and activated in muscular tissues, while no activation of c-Met was observed in tissues of the liver and kidney in comparison with those of non-administration group. It means that HGF provides an effect in muscular tissues to which it is administered, while it provides no effect in the liver and kidney.
Since the effect of HGF was confirmed as described above, the inventors studied practically an efficacy of HGF by intramuscular administration by using an animal model of arteriosclerosis obliterans (ASO). Specifically, by using rat model of hindlimb ischemia, it was studied how ischemic damages of the hindlimb could be improved by the above-described intramuscular administration of HGF. As a method of study, a surface temperature of skin of the hindlimb was measured by using a thermography, and effectiveness of the intramuscular administration was confirmed by using, as an index, inhibition of reduction of the surface temperature of skin. As a result, it was found that reduction of the surface temperature of skin in the hindlimb ischemia was suppressed by the intramuscular administration. It shows that the blood circulation in the hindlimb ischemia was improved by angiogenic effect of HGF, and reduction of the temperature was accordingly suppressed.
Thus, the effectiveness of intramuscular administration of HGF to ASO models was proved for the first time.
The inventors further studied the I/N ratio (%) (blood pressure in an ischemic limb/blood pressure in a normal limb×100) by the intramuscular administration, using rat ASO model of hindlimb ischemia and rabbit ASO model of hindlimb ischemia. As a result, significant improvement of the I/N ratio was observed at a low dose and a low administration frequency. It shows that the blood circulation in the ischemic limb was improved by the angiogenic effect of HGF. Further, by an angiographic study of the rabbit model, an increase rate of the angiographic score was significantly higher in the HGF administration group. It shows that development of collateral vessels was accelerated by the effect of HGF. By also studying arthrosclerosis in the ischemic limb, presence or absence of necrosis and autoamputation of the toe and ulceration in the crus of ischemic limb, tendency of improvement by the intramuscular administration of HGF was observed.
Thus, it was proved that intramuscular administration of HGF was very effective to ischemic diseases.
Confirmation of the effectiveness by using the thermography as the results of the experiments means that the intramuscular administration of HGF is also effective for clinical patients with ASO, particularly, those with cold sense in an ischemic limb as an subjective symptom from a minor stage, and confirmation of the recovery accelerating effect by the I/N ratio and angiography and such improving effect as relief of symptoms in the ischemic limb show that treatment by the intramuscular administration of HGF is effective to patients with ASO of an early to major stages. It enables treatment of ASO in a minor stage before it progresses to a major stage of ASO that requires amputation of a hindlimb due to ischemia, and preparations for the intramuscular administration according to the invention is capable of providing an epoch-making treatment for a lot of patients with ASO of minor stage that cannot be effectively treated today, enabling prohibition of progress of symptoms, improvement thereof and recovery therefrom. Also, they provides a drug providing an efficacy at a low dose, and less affecting other organs except an affected region in comparison with those for intravenous administration.
It was also found that HGF preparations preferably contain no proteoglycans constituting such extracellular matrix as chondroitin sulfate and protein carriers containing them as an ingredient in order to further promote such effect of HGF as described above. In other words, as a result of studying an effect of containment of chondroitin sulfate on release of HGF from a minipellet that was prepared by using collagen as a ground substance, it was found that HGF was released at a higher rate from a minipellet containing HGF with chondroitin sulfate in comparison with that with alanine instead of chondroitin sulfate. It shows that HGF is released at a lower rate in HGF preparations containing no proteoglycans that constitute such extracellular matrix as chondroitin sulfate. It means that HGF preparations containing no proteoglycans that constitute such extracellular matrix as chondroitin sulfate can be more easily retained in local tissues to which it is administered, and transportation and distribution to other organs of the body through the intermediary of the blood can be reduced.